The study of steady-state concentrations of internal solutes of mitochondria by rapid centrifugal transfer to a fixation medium

Abstract

Rapid isolation of rat-liver mitochondria from their suspending medium was achieved by centrifuging them into a layer of silicon fluid. A high-density aqueous "fixative" beneath the silicone stabilized labile phosphate esters when these were to be measured. In the absence of this fixative 19% of the mitochondrial acid-soluble organic phosphate was hydrolysed in 5 min. at room temperature. In this procedure the centrifuge tubes were cooled in a solid carbon dixoide-ethanol bath, the frozen aqueous supernatant fluid was removed, the silicone poured off, and residual silicone removed from the lower layer by washing with hexane at low temperature. The volume of extra-particulate fluid in the mitochondrial pellets as estimated from the distribution of radioactive polyglucose between pellet and medium represented 19-22% of the total water of mitochondrial pellets isolated from 0.25 [image] sucrose solutions at 0[degree]. Mitochondria were permeable to sucrose. The internal concentration of sucrose in mitochondria isolated from 0.25 [image] sucrose solutions at 0[degree] was about 60% of that in the medium. This was tentatively taken to mean that only 60% of the mitochondrial water was permeable to sucrose. The percentage dry weight of mitochondrial pellets isolated from 0.25 [image] sucrose at 0[degree] was 33.9. The presence of polyglucose in the external medium increased the value consistently to 35.3. The concentrations (millimolal) of K, orthophosphate and organic phosphate in the iriternal water of mitochondria isolated from 0.25 [image] sucrose at 0[degree] were about 50, 7 and 40. The use of other salt-poor isolation and suspending media did not result in higher values for these components. Increase in the toncentration of sodium chloride and potassium chloride in the various suspending media in each case by about 0.04 molal caused roughly the same increase in the concentration of these salts in the mitochondrial water. Thus these added substances appear to be passively distributed between mitochondria and medium, and the "bound" ions, in particular K, remain fixed within the mitochondria. The addition of adenine nucleotides and of 2:4-dinitro-phenol to the suspending medium at 0[degree] caused changes in the mitochondrial phosphates and K.